The accumulation of reactive oxygen species (ROS) in the organism, unless counterbalanced by antioxidants produced naturally by the body or taken in through the diet, can cause oxidative damage to DNA and cellular membranes under “oxidative stress” conditions eventually giving rise to certain human diseases, especially cardiovascular disease and some types of cancer. In this context, the measurement of antioxidant capacity of food and biological samples through development of selective and sensitive new techniques has recently gained importance.
Various spectroscopic techniques have been developed for measuring the total antioxidant capacity/activity of pure antioxidant compounds, biological fluids, food extracts, and their various components. These solution-based assays can be broadly classified as electron transfer (ET)-based assays (e.g., CUPRAC, Folin, ABTS/TEAC, FRAP) and hydrogen atom transfer (HAT)-based assays (e.g., ORAC, TRAP). In some cases, these two mechanisms are not differentiated with distinct boundaries. In fact, most non-enzymatic antioxidant activity (e.g., scavenging of free radicals, inhibition of lipid peroxidation, etc.) is mediated by redox reactions.
HAT-based assays measure the capability of an antioxidant to quench free radicals (generally peroxyl radicals) by H-atom donation. Since both the fluorescent probe and antioxidants react with peroxyl radical (ROO) in HAT-based assays, antioxidant activity can be determined from competition kinetics by recording the fluorescence decay curve of the probe in the absence and presence of antioxidants, and integrating the area under these curves to take the difference. HAT-based assays basically include oxygen radical absorbance capacity (ORAC), and total peroxyl radical-trapping antioxidant parameter (TRAP) assays using R-phycoerythrin as the fluorescent probe.
In most ET-based assays, the antioxidant action is simulated with a suitable redox-potential probe, i.e., the antioxidants react with a fluorescent or colored probe (oxidizing agent) instead of peroxyl radicals. Spectrophotometric ET-based assays measure the capacity of an antioxidant in the reduction of a chromogenic oxidant, which changes color when reduced. The degree of color change (either an increase or decrease of absorbance at a given wavelength) is correlated to the concentration of antioxidants in the sample. ABTS/TEAC (trolox-equivalent antioxidant capacity) and DPPH are decolorization assays, whereas in Folin total phenolics, FRAP (ferric reducing antioxidant power) and CUPRAC (cupric reducing antioxidant capacity) assays, there is an increase in absorbance at a prespecified wavelength as the antioxidant reacts with the chromogenic reagent (i.e., in the latter two methods, the lower oxidation states of iron and copper, namely Fe(II) and Cu(I), respectively, emerging as a result of the redox reaction with antioxidants form charge-transfer complexes with the ligands). These assays generally set a fixed time for the concerned redox reaction, and measure thermodynamic conversion (i.e., reduction of the colored species) during that period.
ET-based antioxidant assays generally suffer from reproducibility problems, and those assays performed in solution strictly necessitate accurate measurement and control of reagent and sample volumes, pH, reaction conditions, etc. for standardization of assay protocols. This can be difficult in the existing art, particularly if attempting low cost, high volume, low operator skill applications.
Linearity of responses over a reasonable concentration range together with the additivity of total antioxidant capacity (TAC) values for constituents of complex mixtures would be very desirable for meaningful comparison of TAC value of different food or biofluid samples found with the aid of a sensor. To date, these objectives have not been achieved in a reproducible, low cost and easy to use manner.
There remains a need for sensors that can be particularly suited to rapid and low-cost screening applications evaluation of foods and/or biofluids with sensitivity and precision as well as can solve the linearity problems of previous electrochemical sensors. Other unresolved needs likewise exist.